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• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a class of proteoglycans having fucosylated acidic glyca side chains bound to a protein backbone which have been found to stimulate selectively proliferation of natural killer (NK) cells and/or ⁇ T cells. They are useful as immunostimulants, e.g., in the treatment of cancer and viral infections.
• NK natural killer
• the proteoglycans of the invention are produced by proliferating cells, for example by sponge cells, sea urchin cells, and, in the case of higher animals (including humans), by embryonic cells and tumor cells.
• the compounds are large (ca. 5000 to 30,000 kD) extracellular or membrane-bound molecules having a protein backbone which is glycosylated with acidic glycan chains having an unusual polysaccaride sequence containing internal fucose.
• the structure of the acidic glycan side chains of the proteoglycan isolated from the marine sponge Microciona prolifera has been partially characterized (SpiUmann, et al., J. Biol.
• Microciona or family Mycalidae (e.g., of the genus Mycale), or the order Halichondrida, family Halichondridae (e.g., of the genus Halichondria), or the order Hadromerida, family Clionidae (e.g., of the genus Cliona), or the order Haplosclerida, family Haliclonidae (e.g., of the genus Haliclona),
• the hyperproliferating cells In a pathogenic case, where the hyperproliferating cells are not destroyed in this manner, it is believed that although the hyperproliferating cells produce these acidic glycans, they shed them or present them in monovalent form or other nonstimulatory or inhibitory form, thereby evading detection and destruction by NK cells and/or ⁇ T cells specific for such acidic glycans.
• Application of the compounds of the invention stimulates NK cells and/or ⁇ T cells specific for such cancer cells, thereby leading to their destruction.
• the compounds of the invention are useful for stimulating NK cells and/or ⁇ T cells against viral or retroviral infections.
• the compounds of the invention are useful for inhibiting the activation of NK cells and/or ⁇ T cells, thereby finding utility as immunosuppressants.
• the compounds of the invention are selective in their action, in that particular compounds of the invention stimulate only particular clones or subpopulations of NK cells or ⁇ T cells. No significant stimulation of B cells or ⁇ T cells is observed, so undesirable immunostimulation, e.g., an allergenic or autoimmune response, is avoided. Despite this selectivity, all humans tested, from a variety of ethnic and racial groups, have cell populations capable of being significantly stimulated by the compounds of the invention. Compounds having the glycan structures of the class described herein are found in a wide variety of hyperproliferating cells from sponges to human tumors, thus the basic structure of the compounds is highly conserved.
• compounds of the class described herein act as signals for stimulating the body's defenses against unwanted proliferation of cancerous or infected cells, and that cancers or resistant viral infections may arise when, as described above, these compounds are secreted in nonstimulatory form.
• compounds of the invention isolated from those of the genus Microciona are more effective in stimulating NK cells, as described in example 1 below, whereas compounds isolated from the genus Halichondria are more effective in stimulating ⁇ T cells, as described in example 9, thus selectivity among cell types receptive to this stimulation is also possible.
• the invention thus provides
• proteoglycans and acidic glycans, and/or fragment(s) thereof preferably proteoglycans, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of all the phyla and preferably from an organism of the phylum Porifera, e.g., as described above, especially of the genera Microciona and/or Halichondria and/or Mycale and/or Cliona and/or from an organism of the phylum Echinodermata especially of the genus Lytechinus for use as a pharmaceutical or therapeutic agent in vivo or for ex vivo therapy ; and pharmaceutical compositions comprising such compounds in combination with a pharmaceutically acceptable carrier or diluent.
• proteoglycans e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of all the phyla and preferably from an organism of the phylum Porifera, e.g., as described above
• Novel fucose-containing acidic glycans capable of being isolated from a sea urchin of the genus Lytechinus
• a Fucose-containing acidic glycan for use as a pharmaceutical or therapeutic agent in vivo or for ex vivo therapy ; and pharmaceutical compositions comprising such compounds in combination with a pharmaceutically acceptable carrier or diluent ; and capable of binding to monoclonal antibodies of the type of these named "Block 2" and described in the reference ".Misevic, et al., J. Biol. Chem. (1993) 268 : 4922-4929,
• a method of stimulating the proliferation of mammalian, e.g., human, NK cells and/or ⁇ T cells comprising contacting said cells with a compound of the invention (a fucose-containing proteoglycan and acidic glycan, and/or fragment thereof, preferably a proteoglycan and/or fragment(s) thereof, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of the phylum Porifera, or Echinodermata e.g., as described above, especially of the genera Microciona and/or Halichondria and/or Mycale and/or Cliona, and/or of the phylum Echinodermata especially of the genus Lytechinus, in an ex vivo setting or in vivo, e.g., as a vaccine; or a method of treating cancer (e.g., preventing or inhibiting onset, growth, or metastasis of a tumor), or .
• a viral or retroviral infection in a mammal, e.g., man; comprising administering a pharmaceutically effective amount of a compound of the invention to a patient in need of such treatment; or the use of a compound of the invention in the manufacture of a medicament for treatment or prevention of cancer or viral or retroviral infections.
• a fucose-containing proteoglycan or acidic glycan, or fragment thereof preferably a proteoglycan and/or fragment(s) thereof, e.g., isolated or capable of being isolated from embryonic or neoplastic tissue or from an organism of the phylum Porifera and/or Echinodermata, e.g., as described above, especially of the genera Microciona and/or Halichondria, and/or Mycale and/or Cliona for the phylum Porifera, especially of the genus Lytechinus for the phylum Echinodermata for ex vivo stimulation of proliferation of NK cells and/or ⁇ T cells.
• a method for screening for or detecting an immunosuppressive (e.g., NK cell and/or ⁇ T cell inhibitory) compound comprising measuring proliferation of NK cells and/or ⁇ T cells in a system containing an NK cell or ⁇ T cell stimulatory concentration of a compound of the invention in the presence and absence of a test compound; and compounds identified using such a method.
• an immunosuppressive e.g., NK cell and/or ⁇ T cell inhibitory
• a gene coding for a protein capable of post-translational glycosylation to form the proteoglycan of the invention vectors containing such a gene, and transformed cells, especially (i) production cells, e.g., sponge cells, incorporating such a gene for use in producing the desired proteoglycan at enhanced levels or (ii) cancer cells removed from a patient, transformed with the gene so as to express the proteoglycan in stimulatory form, irradiated, and reintroduced into the patient.
• the gene for Microciona proteoglycan can be isolated, for example, using oligonucleotide probes of a cDNA library based on the disclosed amino acid sequences.
• Appropriate dosages of the compounds of the invention will of course vary, e.g. depending on the condition to be treated (for example the disease type or the nature of resistance), the effect desired, and the mode of administration. In general however satisfactory results are obtained on administration orally, rectally, nasally, topically, or parenterally, e.g. intravenously, for example by i.v. drip or infusion, at dosages on the order of from 0.01 to 2.5 up to 5 mg kg, e.g. on the order of from 0.05 or 0.1 up to 1.0 mg/kg. Suitable dosages for patients are thus on the order of from 0.5 to 125 up to 250 mg i.v., e.g. on the order of from 2.5 to 50 mg i.v.. Pharmaceutical compositions of the invention may be manufactured in conventional manner, in a suitable aqueous carrier, for example steril buffered physiological saline.
• a suitable aqueous carrier for example steril buffered physiological saline.
• suitable amount e.g., at least 10 ml
• peripheral bloo mononuclear cells are isolated from the blood, placed in a complete medium in the presenc of a stimulatory concentration of a compound of the invention, e.g., 10-500 ⁇ g/ml, ca. 100 ⁇ g/ml, optionally in the presence of IL-2, and the culture is maintained until a significan increase in the population of the desired cell type is observed, e.g., ca. 2-4 weeks.
• the cells are isolated from the medium, placed in a injection solution, e.g., sterile buffered physiological saline or plasma, and injected back int the patient.
• a injection solution e.g., sterile buffered physiological saline or plasma
• the compound of the invention for this use can, for example, be a proteoglyca or acidic glycan derived from a marine sponge as described in the examples, but may also b a proteoglycan, acidic glycan or fragment thereof isolated from a culture of the cancerous cells to be treated.
• the compounds can be useful notably as pharmaceuticals, particularly as immunostimulants, e.g. in the treatment of cancer and viral infections.
• EXAMPLE 1 Preparation of proteoglycan and acidic glycans from Microciona prolifera
• Fresh marine sponges (Microciona prolifera) collected from the Cape Cod area (USA) are rinsed with 0.5M NaCl, 0.18g/l NaHCO 3 (buffer A) and cut into cubes lxl cm.
• the cubes are incubated in the buffer A (50% suspension) for 12h at +4°C under gentle rotation. After filtration of the sponge cubes suspension through cheese cloth, the cubes were two more times extracted with the buffer A using the same incubation conditions.
• the supernatants are either combined or separately centrifuged at 3000 x g for 30 min at +4°C, and the obtained supernatant is again centrifuged at 12,000 x g for 40 min at +4°C.
• CaCl 2 is added to the supernatant to a concentration of 20mM. After 2-12 h gentle shaking at room temperature, the precipitated proteoglycan is centrifuged at 3000 x g for 20 min at room temperature. The pelleted proteoglycan is dissolved in at lest 20 volumes of 0.5 M NaCl, 2mM CaCl 2 , 20mM Tris pH 7.4 (buffer B) and centrifuged at 10,000 x g for 30 min at +4°C to remove insoluble material.
• circle portion dissociates from arms in aqueous salt solutions containing less then lmM CaCl 2 or in the presence of EDTA.
• Ca 2+ binding determined by flame ionization spectrometry binds ca. 7000 moles of Ca 2 7mole of proteoglycan at 2mM CaCl 2 and ca. 70,000 moles of Ca 2 7mole o proteoglycan at 20mM CaCl 2 .
• Dissociation fingerprinting Dissociation of proteoglycan by 1%SDS at 100°C gave nine fragments ranging from 38 - 1500 kD on a 5-20% linear gradient polyacrylamide gel after electrophoresis. These fragments had apparent molecular masses of ca. 1500 kD, 500 kD, 250 kD, 150 kD, 148 kD, 135 kD, 108 kD, 70 kD, and 38 kD.
• EDTA and heating at 80°C produced fragments of Mr 1500 x 10 3 , 250 x 10 3 on gel filtration chromatography. Trypsin digestion produced fragments of Mr 124 x 10 3 , 70 x 10 3 , 27 x 10 3 , 10 x 10 3 on gel filtration chromatography.
• Table I shows approximate amino acid (measured by HPLC pico-tag) and approximate total sugar composition (measured by gas chromatography after methanolysis, reacetylation and silylation) :
• This proteoglycan consists of approximatively 36 % by weight proteins and 64 % by weight carbohydrates.
• Standard deviation is less then 20% of each value.
• Asx signifies Asn or Asp;
• Glx signifies
• Glu or Gin It is also noted that apparant amounts of lie and Leu are somewhat variable depending on the preparation.
• the amount of uronic acid determined colormetrically is usually 2 times higher then the amount determined by gas chromatography. SO 4 " was determined by HPLC ion chromatography after hydrolysis of PG.
• N-terminal sequence of the backbone of the molecule is as follows:
• Frozen proteoglycan as obtained above is extracted with water/methanol/chloroform 3/8/4 V/V/V, and the nonlipid fraction was pelleted by centrifugation at 4000 x g for 15 min at +4°C. This extraction is repeated and the pellet is dried under a vacuum. The pellet is wetted in ethanol and resuspended in 0.1 M Tris pH 8, ImM CaCl 2 and 100-200 ⁇ g Pronase
• the glycans are passed through Dowex AG 50W-X8 column in H+ form (Bio-Rad) eluted with water, nonbound glycans are immediately neutralized and electrophoresed on a 5-20% or 10-40% linear polyacrylamide gradient gels (Tris/borate- EDTA), and separated acidic glycans of Mr 200 x 10 3 are eluted from gels. (Optionally, the acidic glycans can be sepatated by gel filtration rather than electrophoresis).
• the isolated acidic glycan molecules are desalted using P-2 column (Bio-Rad) eluted with lOmM pyridine acetate pH 5, lyophilized and stored at -20°C.
• the acidic glycan fraction is comprised of two major glycans of apparent molecular mass determined by gel electrophoresis using glycosaminoglycan standards of ca. 200 kD and 6 kD.
• the glycans have the foUowing molar composition (expressed as moles of monosaccharide units / mole of glycan), as determined by gas chromatography, as shown in Table II:
• Standard deviation is less then 20% of each value.
• Per mole of proteoglycan there are 20 moles of the 200 kD glycan and 1000 moles of the 6 kD glycan.
• the glycans are not digestible with Chondroitinase A, B, C, Heparinase, Heparitinase, Hyaluronidase or Keratinase. They are soluble in aqueous solutions and do not form gels in 6mM CaCl 2 salt solutions. At higher concentrations, e.g. > 1 mg/ml water, they will undergo hydrolysis at room temperature.
• proteoglycan Extraction of proteoglycan from Halichondria panicea and isolation of acidic glycans from Halichondria panicea proteoglycan is performed as described in example 1 for Microciona prolifera.
• the proteoglycan thus obtained has the following characteristics:
• This proteoglycan consists of approximately 79 % protein and 21 % carbohydrate by weight. It has an approximate amino acid composition (as measured by HPLC pico-tag) and approximate total sugar composition (as measured by gas chromatography) as shown in Table UI : Table III
• Isolation of acidic glycans from this proteoglycan in the manner described in example 1 gives seven glycans having apparent molecular mass determined by gel electrophoresis using glycosaminoglycan standards of ca. > 1000 kD, 600 kD, 160 kD, 150 kD, 110 kD, 82, kD, and 50 kD.
• EXAMPLE 3 Preparation of proteoglycans and acidic glycans from Mycale lingua.
• Mycale lingua proteoglycan is performed as described in example 1 for Microciona prolifera.
• the proteoglycan thus obtained has the following characteristics:
• This proteoglycan consists of approximately 58% protein and 42% carbohydrate by weight. It has an approximate amino acid composition (as measured by HPLC pico-tag) and approximate total sugar composition (as measured by gas chromatography) as shown in Table IV : Table IV Amino acid composition and carbohydrate composition
• Glu or Gin It is also noted that apparant amounts of He and Leu are somewhat variable depending on the preparation. The amount of uronic acid determined colormetrically is usually 2 times higher then the amount determined by gas chromatography. SO 4 " as determined by HPLC ion chromatography after hydrolysis of PG.
• CPGl consists of approximately 26 % protein and 74 % carbohydrate by weight
• CPG2 consists of approximately 32 % protein and 68 % carbohydrate by weight. They have an approximate amino acid composition (as measured by HPLC pico-tag) and approximate total sugar composition (as measured by gas chromatography) as shown in Table V :
• Glu or Gin It is also noted that apparant amounts of He and Leu are somewhat variabl depending on the preparation.
• the amount of uronic acid determined colorimetrically i usually 2 times higher then the amount determined by gas chromatography. SO 4 " wa determined by HPLC ion chromatography after hydrolysis of PG.
• Lytechinus pictus sea urchin eggs and/or embryos (from 2 cell stage to plutes stage) were washed with sterile sea water and pelleted embryos were extracted with water/methanol/chloroform 3/8/4 V/V/V, and the nonlipid fraction was pelleted by centrifugation at 4000 x g for 15 min at +4°C. This extraction is repeated and the pellet is dried under a vacuum.
• the pellet is wetted in ethanol and resuspended in 0.1 M Tris pH 8, ImM CaCl 2 and 100-200 ⁇ g Pronase (Calbiochem) (preincubated for 30 min at 60°C in 0.1M Tris pH 8, ImM CaCl,per l-2mg dried powder material), and the pellet is digested at 60°C for three days. Two more equivalent portions of preincubated pronase are added at 24 h intervals. DNAse I is then added (30 ⁇ g) and incubation is continued at 37°C in the presence of lOmM MgCl 2 .
• the digested sample is then treated again with pronase and chromatographed through G-25 Sephadex (Pharmacia) column eluted with lOmM pyridine acetate pH 5, void volume fractions are collected and lyophilized, and the glycans thus obtained are dissolved in 50mM NaOH in the presence of IM NaHBO 4 and incubated at 45°C for 16h (NaOH treatment may also be omitted).
• the glycans are passed through Dowex AG 50W-X8 column in H+ form (Bio-Rad) eluted with water, nonbound glycans are immediately neutralized and electrophoresed on a 5-20% or 10-40% linear polyacrylamide gradient gels (Tris/borate-EDTA), and separated acidic glycans of Mr 200 x 10 3 are eluted from gels.
• the acidic glycans can be sepatated by gel filtration rather than electrophoresis).
• the isolated acidic glycan molecules are desalted using P-2 column (Bio-Rad) eluted with lOmM pyridine acetate pH 5, lyophilized and purified by affinity chromatography with the Block 2 monoclonal antibodies of ref Misevic et al mentioned above stored at -20°C.
• Standard deviation is less then 20% of each value.
• the amount of uronic acid determined colormetrically is usually 2 times higher then the amount determined by gas chromatography. SO " was determined by HPLC ion chromatography after hydrolysis of PG.
• EXAMPLE 6 Ex vivo stimulation of human NK cells proliferation by Microciona prolifera proteoglycan and by its acidic glycans
• PBMC peripheral blood mononuclear cell
• NK cells population CD 16 and CD 56 positive
• CD3, TCR ⁇
• TCR ⁇ , CD4, CD8, CD20 and CD14 negative increased from 1-5 % to 30-80 % of the total PBMC, whereas untreated controls remained at a level of 1-5 % NK cells.
• Specific stimulation of NK cells proliferation by glycans was confirmed by 3 H thymidine incorporation only in isolated clones of NK cells and not ⁇ T cells isolated from the same
• EXAMPLE 7 Ex vivo stimulation of human NK cells proliferation by Mycale lingua an Cliona celata proteoglycans and by its acidic glycans was similar to Microciona prolifera proteoglycan.
• EXAMPLE 8 Ex vivo stimulation of human NK cells proliferation by Lytechinus pictu acidic glycan with 580 kD was similar to Microciona prolifera proteoglycan.
• EXAMPLE 9 Stimulation of human ⁇ T cells proliferation (ex vivo) by Microcion prolifera proteoglycan, Halichondria panicea proteoglycan and /or their acidic glycans
• Microciona prolifera acidic glycans stimulate only one subpopulation of ⁇ T cells via T cell receptor with an increase from 5% to 20%.
• Halichondria panicea proteoglycan and its acidic glycans stimulate a different population of ⁇ T cells from 5% to 70%.
• EXAMPLE 10 Anti-tumorogenic and anti-metastatic activity of proteoglycans from Microciona prolifera (in vivo)
• mice Seven C-57 black mice are injected i.p. with 300 ⁇ g proteoglycan from Microciona prolifera/200 ⁇ 0.2M NaCl, 2mM CaCU, 20mM Tris pH 7.4/animal, every day for five days. At day five, animals are injected with 2.5 X 10 4 B-16 melanoma cells per animal. Animals are immunized for five more days with proteoglycan as described above. The appearance of tumor, tumor growth, survival of animals and appearance of metastasis are observed in immunized animals and compared with control animals injected with buffer. Control animals which have not received proteoglycan all exhibit marked melanoma growth followed by metastasis.
• EXAMPLE 11 Anti-tumorogenic and anti-metastatic activity of proteoglycans and their acidic glycans from Halichondria panicea Mycale lingua, Cliona celata and Lytechinus pictus were similar to to Microciona prolifera proteoglycan (in vivo).
• EXAMPLE 12 Cloning and expression of gene for proteoglycans from Microciona prolifera
• Proteoglycan (PG) cDNA is isolated from a random-primed cDNA library created using poly(A) + RNA from Microciona prolifera cells. This cDNA library is screened using the N-terminal amino acid sequence of PG described in example 1 above by colony hybridization techniques, i.e., expressing the library in an expression system, preferably E.
• coli lysing the colonies, e.g., on nitrocellulose filters, denaturing their DNA in situ and fixing it on the filter, hybridizing with labeled, preferably radiolabeled, oligonucleotide probes of at least 30 base pairs having cDNA base sequences corresponding to all or a portion of the N-terminal sequence of PG, identifying hybridized colonies, and retrieving the corresponding vectors from the library, using chromosome walking techniques if necessary to isolate and characterize one or more cDNA fragments containing one or more regions coding for glycosylation sites for N-linked glycans. (Note that the cDNA is repetitive, so it is not necessary to clone, isolate and characterize the entire sequence).
• a suitable expression system preferably a eukaryotic system, most preferably a sponge.
• the PG is isolated from the sponge or from the culture medium of the expression system, e.g., using the procedures outlined above.

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